Process for concentration of neovitamin a



Patented May 15 195i PROCESS FOR CONCENTRATION OF NEOVITAMIN A Charles" ,Rqbesdny Rochester, N.. Y assignor,

bymesneassignin nts, Eastman Kodak Come pan'yi'Bo'chestei", NI YL, acorporation of New Jersey N Drawing. Continuatijo n of application Serial No'ig 555,405, Siieptemberf 22; 19,44. -,This application December 12, 1947, Serial No. 791,449

3 Claims." (o1; 260- 617) 1 This application is a continuation of Serial No. 555,405, filed September 22, 1944, now abandoned. This invention relates, to" improv d rocedure for prpa'ration 'of' products having'yitainin' ac tivity and to improved vitamin A products? Anobject of theinventionis tdpr'ovifglefa new vitamin A product havingimproved properties. Another object is to provide improved procedure for preparing new 'vitamin' A-active substances. Another object is-to iniprove the stateof the art. Other objects will appear hereinafter.

These and other objects are accomplished by my-inventionwhich includes separating "substantially all ofthe'ordinary or knowmvitamin A from' the nonesaponifiablematterof a fish oil,"

andthensubjecting the residuefreed of known vitamin-A toa treatmentfor" removal "of'a" substance which has substantially differentproper: ties from known vitamin A, but whichbiihibits' 20 high vitamin A activity; V In tli'e 'follo'wing' examples and 'desc'riptio" have'set' forth severaij [of l the pre en eu einbo ments of my invention; but it is to be underst that these are given for the purpose of illustration and'not in'limitat-io'n thereof."

The non-saponifiable matter may be obtained from thefish oil in any desiredmanner: Like= wise the non-saponifiable"matter'may befireed of its'ordinary vitamin A alcohol content in' any of the known Ways. Thus," for instance, the non-saponifiable matter may be obtained by direct saponification of the fish oil. A more economical methodis first to subject the fishbil to molecular distillation to separate a vitamin A concentrate-as a distillate and then 'tosap'onify this distillate to' obtain' the L non sapo'nifiable matter. The removal of the krioWn vitamin A from the non-sapo'nifiable matterniay be" ad complishedf for example, by crystallizing the vitamin A alcohol "from'a solution thereof "ina solvent "such as" ethyl formatef "solution is usually cooled to "a low temperature suclf as 2 separate the" new vitamin A active" sttsafies (hereinafter referred, to as neo-vitamin A). The two methods which'have been found best for this procedure "are chromatographic "adsorption and crystallization from solvents or a combination thereof. Suitable adsorbents aref zeolites" such as Doucil. (a manufactured so dium aluminum silicate), silica" gel, activated aluminum oxide, calciumhydroxide zinc carbo'nate, activated] carbonpetct The adsorbent may be contacted with the residue containing the new vitamin A active substance in any desired manner. Thus, a solution of the residue} in a suitable solvent may be passed through an adsorption column containing the adsorbent. The neo-vitamin A becomes adsorbed thereon and is then eluted from the column witha solvent. Also, it is feasible merely tojfstifi the adsorbent with the solvent solution Vitamin A amaha boniiit is thri treated to "cema amg'whe. neo-vitamin A residue, separate the adsorbent, and then elute the nee-vitamin ATthelQfIQmT; Crystallization may be accomplishedflby"dissolving the residue in a suitable solvent and cooling to a low temperature, or if desired, the neo,-v itamin;A contained in theresi du can b rea tediiwith a derivative .to form; an. ester having: crystallizable, properties, and these esters then may be crystallized from solution by cooling; The crystalline. esters .thus obtained may be directly used, or it may be.ad-. vantageous to" saponify them'iand thus? obtain the purified) nee-vitamin 'A.. alcohol; which. then can beseparated from the saponified mixture. and crystallized from a, solvent. .as described above. 'The factTthat nee-vitamin A forms "a mono; ester clearly shows that it! is an alcohol.

It has been discovered that fish body and liver oils as a class contain neo-vitamin A and may be used as'a starting'material'. If the oil is low in vitamin A content, 11-. shouldprefe'rably be concentrated by high-vacuum unobstructed path distillaf" before 'saponificat Examples of suitable "oils "are" oo'd"1iver, sardine, polla'ck' liver and shark liver oils?" The propertiesof neo -yitaminuA .are comred. wi h P i??? .kn wn. vit co Table e-same ester; i e. ngg yitamin A esters with vitamins. esters, in Tabi'ii?" Table I M I t Recoverg of vlita- Extinc- P. Re a ive min in o ive gi f tion 23 Anthra- Rate of oil 501. after 1 hr. Crystalline Structure M. P eng 9 elficient p quinoue Dehydraexposure to air absolptwn 1% g Carboxtron by at 55 C. By maxlmum 1 cm. Que ylate A10. 1101 Baxter Rocker Equipment Degrees M 121-122 Per cent Vit. A Yellow prisms 62-64 325 1, 750 Strong Yellow. Fast 65 N eo Vit. A Light yellow feathery 134l36 crystals 59-60 328 1, 645 Weak Red. Slow V 90 (Ext. coefi. and abs. max. were measured on ethyl alcohol solutions of the vitamin A compounds by the Hilger quartz spectograph, model E-498 with a Spekker U. V. photometer. Light source-Tungsten steel spark.)

Table II 17 Relative M. P. L Value gg iggifi 1 m. rate of Crystalline Structure Azoyl of A 17 of antimony dehydra- Ester Ester E (328) trichloride tion In 0 Blue "color alcohol Degrees Vit. A Azoate Red, Orange Prisms. 79-80 2, 340 1, 495 2, 330 Fast. Neo Vit. A Azoate OiiaInggfr Rust feathery 94-96 2, 100 1, 460 2, 230 Slow.

ee es.

(The L value is a measure of the intensity of the antimony trichloride blue color as measured by Evelyn 1 cm (620 m photoelectric colorimeter and is analogous to E Example (A) Saponificatzon.-113 grams of a vitamin A ester concentrate prepared by high-vaccum distillation of ling cod liver oil (E (1%, 1 om.)=500) at 328 m was saponified for minutes at 70 C. by 318 cc. of 2 N alcoholic KOH, in an atmosphere of nitrogen. The non-saponifiable matter was obtained by diluting the recolumn mm. diameter) of Doucil (a sodium aluminum silicate) (250 gms.) and washed with additional petroleum ether until a yelloworange zone reached the bottom of the column. The column was removed in four equal sections which were separately eluted with ethyl ether containing 5% methyl alcohol. Thus, there was obtained five fractions as shown in Table III.

Table III Anpydro Per Cent Fraction Blank Wt. Ei'fi 328 m) A Neo V E (392 m Vitamin A Orig. Filtrate I 2. 5 Sec. 1 of Column (Bottom) 8. 7 1, 040 227 1 88 Sec. 2 of Column 3. 5 l, 485 40. 8 40 Sec. 3 of Column 3. l 1, 440 43. l 30 Sec. 4 of Column (Top) 2.1 1, 350

1 Abnormally high because of presence of anhydro vitamin A which interferes with the determination.

action mixture with water and extracting with ether according to the well known procedure. After removing the solvent 42.5 g. ofnon-saponifiable containing vitamin A alcohol (E (1%, 1 cm.) =1110) 328 m was obtained as a dark red viscous oil.

(B) Crystallization to remove vitamin A alcohol-.The non-saponifiable from (A) was dissolved in 300 cc. of ethyl formate and cooled to 35 C. After 18 hours the precipitated sterols (7.05 g.) were filtered. Re-cooling the filtrate to 8() C. caused no more solids to deposit, so the 7 solution was seeded with a few crystals of vitamin A alcohol. In two days the precipitation of vitamin Afwas complete and the crystals were removed by filtration.

(C) Preparation of neo-m'tamin A and aeo benzene carboxyZates.-The filtrate residue from (B) was freed of ethyl formate by distillation under vacuum and the residue (20 grams, E??? (328 m,u=1150)) was dissolved in petroleum ether to make 150 cc. of solution which was chromatographed. on a and ' Eli... 392=24 which contained approximately neo-vitamin A. To this neo-vitamin A concentrate (4.9 gm.) in methylene chloride (3000.) and pyridine (5 cc.) was slowly added azo benzene carboxyl chloride (4.25'gms.) in methylene chloride (30 cc.) After standing 5 hours at room temperature, water (2 cc.) was added and the reaction mixture warmed to 50 to hydrolyze any excess acid chloride. The methylene chloride solution was then poured into 5% H01 (200 cc.) and extracted with ether (300 0a.). The ether extract was washed with 5% HCl to remove pyridine, N/5 KOH, and finally with water to neutrality. The solvent was 75 evaporated after drying by filtration through sodium sulfate yieldinga red oil (85 gm.) Petroleum ether (100 cc.) was added and the solution filtered and washed througha column of Doucil (a sodium aluminum silicate) to remove the azoic anhydride with which the product was contaminated. The petroleum ether filtrate and washings were concentrated to '75 cc. and cooled to 35 C. which caused crystallization of the neo-vitamin A azo benzene carboxylate (3.1 gm.). After two recrystallizations of the ester from petroleum ether at 25, the orange crystals (1.35 gm., II) were dried under vacuum, feathery needles, melting point 94-96% A depression of melting point occurred when mixed with a sample of vitamin A azo benzene carboxylate.

Neo-vitamin A azoate (1 gm., II) was dissolved in 10 cc. of boiling alcohol, and 4 cc. of 4 N alcoholic KOH was added. The mixture was refiuxed for 15 minutes. After pouring into water (50 cc.) and extracting with ether (100 co.) the ether extract was washed with N/ KOH and water, dried over sodium sulfate, filtered and evaporated to yield neo-vitamin A as a viscous yellow oil (.6 gm.) which crystallized from ethyl formate (2 cc.) at 35. The pale yellow needles of neo-vitamin A were filtered and dried under vacuum M. P. 59-60";

When mixed with vitamin A (M. P. 62-64") a depression in the melting point to approximately 54 was observed.

The data on rates of dehydration show that the neo-vitamin A in alcohol form is more stable to acid than known vitamin A and that the ester is more stable to action of alcohols than the same ester of known vitamin A.

The vitamin A azo benzene carboxylate mentioned above was prepared from crystalline vitamin A M. P. 62-64 C. and azo benzene carboxyl chloride using the procedure as described for neovitamin A azo benzene carboxylate. Reddish orange prisms melting point 79.5-80;

Eff, 330 mp=1460 Anthraquinone beta-carboxylate of neo-vitamin A is prepared as follows: To a solution of neo-vitamin A (0.15 g.) in methylene chloride (1 cc.) and pyridine (0.2 cc.) was added a solution of anthraquinon beta-carboxyl chloride (0.15 g.) in benzene (4 00.). After standing at 25 for four hours, the esterification mixture was poured into 5% HCl (15 cc.) and extracted with ether (20 00.). The ether extract was washed with 5% HCl to remove pyridine, N/5 KOH, and finally with water to neutrality. Evaporation of the solvent yielded a red viscous oil 0.2 g.) which crystallized from acetone (2 cc.) at 0. Recrystallization from acetone at 25 yielded red rosette like crystals M. P. 134-136 C.

Substitution of vitamin A for neo-vitamin in the above procedure yielded yellow plate like crystals. M. P. 121-122 C.

Vitamin A has the following structural formula:

Vitamin A It can be seen that the double bond nearest the hydroxyl group enables the molecule to occur in either cis or trans form. The new vitamin A active substance prepared in accordance with the present invention and termed nee-vitamin A has been found to have the following cis configuration:

H36 CH Neowitamin A The corresponding trans isomer is the previously known vitamin A.

While the invention has been described in considerable detail in connection with certain preferred procedures and materials, it will be understood that modifications and variations may be effected therein without departing from the spirit and scope of the invention as it is defined by the appended claims.

What I claim is:

1. The process of preparing a concentrate of neo-vitamin A having greater stability to oxidation than vitamin A and having substantial vitamin A activity, which process comprises saponifying a fish oil, separating the saponified portion of said oil from the unsaponified portion thereof, separating vitamin A alcohol from said unsaponified portion and thereby leaving a residual portion containing neovitamin A, and thereafter separating and concentrating said neovitamin A from said residual portion by subjecting said residual portion to chromatographic adsorption effective to adsorb said neovitamin A, and separately eluting said neovitamin A and thereby obtaining a concentrate of neovitamin A containing a substantially higher percentage of neovitamin A than said fish oil and being substantially free of vitamin A.

2. The process of preparing material characterized by having substantial vitamin A biological activity and having greater stability to oxidation than vitamin A, which process comprises saponifying a fish oil, separating the saponified portion of said fish oil from the unsaponified por tion thereof, separating vitamin A alcohol from said unsaponified portion and thereby leaving a residual portion containing neovitamin A, separating and concentrating said neovitamin A from said residual portion by subjecting said residual portion to chromatographic adsorption effective to adsorb said neovitamin A and separately eluting said adsorbed neovitamin A, esterifying said separated and concentrated neovitamin A, and crystallizing out the resulting esterified neovitamin A.

3. The process of preparing a concentrate of neovitamin A characterized by having substantial vitamin A activity and having greater stability against oxidation than vitamin A, which process comprises saponifying a fish oil, separating the saponified portion of said fish oil from the unsaponified portion thereof, crystallizing vitamin A from said unsaponified portion and thereby leaving a residual portion containing neovitamin A, passing said residual portion through a body of adsorbent and thereby adsorbing said neovitamin A separately from the remainder of said residual portion, separately eluting said adsorbed neovitamin A, and evaporat.

'ing the iesuiting eiuate and thereby recovering a neovitamin A concentrate substantially free of vitamin A.

CHARLES D. ROBESON.

REFERENCES CITED The following references are of record in the file of this patent:

8 UNITED STATES PATENTS 1 OTHER REFERENCES Baxter et aL: Jour. of Biol. Chem, vol. 141, 991-2 (1941). r 

1. THE PROCESS OF PREPARING A CONCENTRATE OF NEO-VITAMIN A HAVING GREATER STABILITY TO OXIDATION THAN VITAMIN A AND HAVING SUBSTANTIAL VITAMIN A ACTIVIATY, WHICH PROCESS COMPRISES SAPONIFYING A FISH OIL, SEPARATING THE SAPONIFIED PORTION OF SAID OIL FROM THE UNSAPONIFIED PORTION THEREOF, SEPARATING VITAMIN A ALCOHOL FORM SAID UNSAPONIFIED PORTION AND THEREBY LEAVING A RESIDUAL PORTION CONTAINING NEOVITAMIN A, AND THEREAFTER SEPARATING AND CONCENTRATING SAID NEOVITAMIN A FROM SAID RESIDUAL PORTION BY SUBJECTING SAID RESIDUAL PORTION TO CHROMATOGRAPHIC ADSORPTION EFFECTIVE TO ADSORB SAID NEOVITAMIN A, AND SEPARATELY ELUTING SAID NEOVITAMIN A AND THEREBY OBTAINING A CONCENTRATE OF NEOVITAMIN A CONTAINING A SUBSTANTIALLY HIGHER PERCENTAGE OF NEOVITAMIN A THAN SAID FISH OIL AND BEING SUBSTANTIALLY FREE OF VITAMIN A. 